Method for ex vivo immunization using heterologous intact bispecific and/or trispecific antibodies

ABSTRACT

According to the invention there is described a method for ex vivo immunization of humans and animals comprising the following steps of:  
     a) isolating autologous tumor cells;  
     b) treating the tumor cells to prevent the survival thereof following reinfusion;  
     c) incubating the thus treated tumor cells with intact heterologous bispecific and/or trisepcific antibodies showing the following properties:  
     α—binding to a T cell;  
     β—binding to at least one antigen on a tumor cell;  
     γ—binding, by their Fc portion (in the case of bispecific antibodies), or by a third specificity (in the case of trispecific antibodies) to Fc receptor-positive cells.

[0001] The invention relates to a method for ex vivo immunization usingheterologous, intact bispecific and/or trispecific antibodies as well asthe use of the products of said method in the prevention and therapy oftumourous diseases and in particular in the induction of an anti-tumourimmunity.

[0002] Despite the progresses in chemotherapy and radiotherapy achievedduring recent years, malignant diseases in humans, for example advancedbreast cancer, still have an extraordinarily unfavourable prognosis.Generally, such diseases are impossible to heal. Therefore, it isnecessary to develop novel treatment strategies. In this respect, greathopes are placed on immunotherapeutic approaches which shall be used toinduce the patient's immune system to reject the tumour. It is wellknown that tumour-associated antigens are present on tumour cells, andthat in principle the immune system is very well able to recognize theseantigens and to attack the malignant cells. However, tumours havedeveloped various strategies which enable them to escape the immunereaction. They achieve this for example by an insufficient presentationof tumour-associated antigens and/or insufficient activation oftumour-specific T cells which are generally present.

[0003] With about 43,000 new cases/year, breast cancer occupies a topposition in the cancer statistics of women in Germany. Less than onethird of the women suffering from lymph node invasion at the time ofdiagnosis survive for 10 years without relapse.

[0004] To date, the immunotherapeutic approaches towards mamma carcinomahave been restricted to methods for unspecific stimulation, such astreatment by BCG or levamisole, and to the use of LAK and NK cells withIL-2 (3, 4). However, the types of immunotherapy employed provided noevidence for a prolongation of life; the treatment by BCG even proved tobe disadvantageous (3). Since the unspecific activation of cells has notbeen very successful also in other types of tumour, attempts were madetowards the induction of a specific immune reaction.

[0005] For example, T cell-redirecting bispecific antibodies were usedin tumour therapy. These antibodies bind with one of their binding armsto a T cell receptor complex and with their other binding arm to atumour-associated antigen on a tumour cell. The resulting activation ofthe T cell and the spatial proximity of the tumour cell leads todestruction of the latter by induction of apoptosis or by cytokins, suchas TNF-α or perforin, respectively.

[0006] The antibodies used in tumour therapy in the prior art weredirectly infused into patients. This type of procedure shows severaldisadvantages:

[0007] it requires high doses of antibodies;

[0008] severe side effects may occur;

[0009] by their tumour binding arm the antibodies may also bind tonormal tissue during in vivo application.

[0010] It is an object of the present invention to provide a novelmethod for the therapy of malignant diseases in humans, in particularwith the objective of achieving an anti-tumour immunity.

[0011] According to the invention, this object has been achieved by themethod characterized in more detail in claim 1. Preferred embodiments ofthe method become clear from the dependent claims.

[0012] The end product of the method of the present invention is atumour cell preparation containing antibodies. This tumour cellpreparation is used in the prevention and treatment of tumorous diseasesby inducing an anti-tumour immunity.

[0013] By using the method of the present invention autologous tumourcells are treated with heterologous bispecific and/or trispecificantibodies, and the tumour cell preparation obtained by the presentmethod is used for reinfusion into the patient or the animals from whomthe autologous tumour cells have been obtained.

[0014] The invention relates further to the use of the method and thetumour cell preparations provided according to the invention in theprevention and therapy of tumourous diseases, in particular in theachievement of an anti-tumour immunity and particularly preferred of along-term immunity.

[0015] The experiments provided in the present invention, particularlyexample 2, show that a long-lasting tumour immunity is provided. Theresults of the experiment performed in mice can be transfered also tohumans. It is expected that a long-term immunity of several years can beprovided by using the present invention. A tumour cell as descibed inthe present invention is every cell which has lost its normal func-tionby one or more mutations or wherein its normal function has beenchanged. Due to these mutations the tumour cells are able to propagatein an uncontrolled manner.

[0016] Tumour immunity according to the present invention is defined byactivating the immune system of the body in an organism against theautologous tumour in such a way that a long-term or even permanentdestruction and/or control of the autologous tumour is achieved.

[0017] According to the invention every kind of tumours falling underthe definition given above can be treated by the present method.Particularly epithelial tumours, adenocarcinomas, colon carcinomas,mamma carcinomas, overial carcinomas, carcinomas of lungs, throat, noseand ear can be treated. Furthermore, preferably non-epithelial tumourslike leukaemias and lymphomas and virus inducted tumours like livertumours or cervix carcinomas can be treated.

[0018] According to the invention, heterologous intact bispecific and/ortrispecific antibodies are used. These antibodies are contacted ex vivowith tumour cells (autologous tumour cells) previously obtained from apatient. To prevent the survival of tumour cells following reinfusion,the tumour cells were treated in a manner known per se, such as byirradiation, prior to contacting with the antibodies. Followingirradiation, the tumour cells are incubated with the intact heterologousbispecific and/or trispecific antibodies. According to the invention,not any antibody may be used but only antibodies which are intact, i.e.having a functional Fc portion, and they must be heterologous in nature,i.e. such antibodies which consist of heavy immunoglobulin chains ofdifferent subclasses (subclass combinations, also fragments) and/ororigin (species).

[0019] These intact heterologous bispecific and/or trispecificantibodies will be selected to further have the following properties:

[0020] α—binding to a T cell;

[0021] β—binding to at least one antigen on a tumour cell;

[0022] γ—binding, by their Fc portion (in the case of bispecificantibodies), or by a third specificity (in the case of trispecificantibodies) to Fc receptor-positive cells.

[0023] In a particularly preferred embodiment of the present inventionthe intact heterologous bispecific and/or trispecific antibodies areselected to be able to activate the Fc receptor-positive cell, andthereby inducing or increasing the expression of cytokins and/orco-stimulatory antigens. The tumour cell preparation obtained includingsaid antibodies is them prepared further for reinfusion. It istransferred for instance in a device suitable for reinfusion.

[0024] In the case of trispecific antibodies, binding to the Fcreceptor-positive cells preferably takes place via the Fc receptor of Fcreceptor-positive cells or also via other antigens on Fcreceptor-positive cells (antigen-presenting cells), such as the mannosereceptor.

[0025] Only the present method and the use of the antibodies describedherein ensures the development of an anti-tumour immunity afterreinfusion of the antibodies into the patient from whom the tumour cellshave previously been obtained. Preferably, reinfusion is carried out ina patient after treatment of the primary tumour, preferably in patientsin a minimal residual disease (MRD) situation. In patients with fewresidual tumour cells but with a high risk of relapse, use of the methodprovided according to the invention will be particularly successful.

[0026] By using the method of the invention, it is possible to avoid thedisadvantages known from the prior art and described in more detailabove.

[0027] The heterologous bispecific and/or trispecific antibodies usefulaccording to the invention are in part known per se, but in part theyare described for the first time in the present application. An examplefor a bsab is antibody anti-CD3 x anti-epcam which is employed inepithelial tumours such as mamma carcinoma.

[0028] According to the invention, two variations of the method may bedistinguished:

[0029] 1. short-term incubation, and

[0030] 2. long-term incubation.

[0031] A short-term incubation is an incubation of the autologous tumourcells with intact heterologous bispecific and/or trispecific antibodiesfor a period of 10 minutes to 5 hours, or 10 minutes to 3 hours, orfurther preferred for a period of about 15 minutes to 2 hours, furtherpreferred for a period of 15 minutes to 1 hour. The tumour cells chargedwith antibodies in this way are then prepared for reinfusion.

[0032] The long-term incubation is an incubation of the autologoustumour cells also for a period of about 10 minutes to 5 hours,preferably for a period of 15 minutes to 2 hours and further preferredfor a period of 15 minutes to 1 hour, so that the autologous tumourcells are charged with antibodies. Subsequently, blood cells of thepatient, preferably mononucleated cells of the peripheral blood(PBMCs=peripheral blood mononucleated cells) are added, and this mixtureis then incubated over a prolonged period, such as 1 to 14 days,preferably 3 to 10 days and further preferred 6 to 10 days.Alternatively, another way of proceeding is contacting the autologoustumour cells directly with the bispecific and/or trispecific antibodiesand with the patient's blood cells, preferably peripheral bloodmononucleated cells. In this way, “priming” of numerous immune cellsagainst the tumour is achieved already ex vivo. Afterwards, these cellsare reinfused into the patient. Long-term incubation also leads tointernalization and degradation of the antibodies.

[0033] Preliminary in vitro results show that immune cells pretreated inthe way described are able to destroy tumour cells without furtheradditon of bispecific and/or trispecific antibodies (cf. Example 1).

[0034] In short-term as well as long-term incubation, the T cells areredirected to the tumour cells by the bispecific and/or trispecificantibodies which are immobilized on the tumour cells; at the same timebinding of Fc receptor-positive cells to the Fc portion of thebispecific and/or trispecific antibody takes place after reinfusion.This leads to activation of Fc receptor-positive cells by their bindingto the Fc portions of immobilized (on the T cell or tumour cell,respectively) intact bispecific antibodies.

[0035] To enhance the success of immunization, the tumour cells treatedwith the antibodies either according to the short-term incubation methodor the long-term incubation method may be administered to the patientnot only once but optionally also several times.

[0036] On the tumour cell, an up-regulation of MHC 1 as well asactivation of the intracellular processing machinery (proteasomecomplex) occurs due to the release of cytokins (such as INF-γ and TNF-α)in direct proximity of the tumour cell. Cytokins are released because ofthe bispecific antibody-mediated activation of T cells and accessorycells (see FIGS. 1 and 3). I.e. by the intact bsab not only tumour cellsare destroyed and phagocytized but indirectly also their anti-tumourimmunity is increased.

[0037] Activation of the Fc receptor-positive cells by the bsab dependson the subclass or subclass combination, respectively, of the bsab. Asdemonstrated in in vitro experiments, for example bsabs of the subclasscombination mouse IgG2a/rat IgG2b are able simultaneously to bind to andactivate Fc receptor-positive cells leading to up-regulation andformation (expression), respectively, of co-stimulatory antigens, suchas CD40, CD80, or CD86, on the cell surface of such cells. In contrast,bsabs of the subclass combination mouse IgG1/IgG2b are able to bind toFc receptor-positive cells (1) but clearly are unable to activate thesecells to a comparable extend (2).

[0038] While the intact bsab at the same time binds to and activates theT cell via one of its binding arms (e.g. to CD3 or CD2), co-stimulatorysignals derived from the Fc receptor-positive cell bound to the Fcportion of the bsab may be transferred to the T cell. I.e. only thecombination of T cell activation via one binding arm of the bsab and theconcomitant transfer of co-stimulatory signals from the Fcreceptor-positive cell to the T cell results in an effective T cellactivation (FIG. 1A). Tumour-specific T cells which have beeninsufficiently activated at the tumour cell and are anergic may also bereactivated according to the ex vivo pretreatment of the invention (FIG.1B).

[0039] A further important aspect in the induction of anti-tumourimmunity is the possibility of phagocytosis, processing and presentationof tumour components by accessory cells (monocytes/macrophages,dendritic cells, and NK—“natural killer”—cells) which have been directedand activated by the bsab. By this classical mechanism of antigenpresentation tumour-specific CD4 cells as well as CD8 positive cells canbe generated. Moreover, tumour-specific CD4 cells play an important rolein the induction of a humoral immune reaction in the context of the T-Bcell cooperation.

[0040] Bispecific and trispecific antibodies are able to bind to the Tcell receptor complex of the T cell by one binding arm and totumour-associated antigens on the tumour cells by the second bindingarm. Thereby, they activate T cells which destroy the tumour cells byreleasing cytokins or apoptosis-mediating mechanisms. Furthermore, inthe context of their activation by bispecific antibodies it is clearlypossible for T cells to recognize tumour-specific antigens via theirreceptor whereby a long-lasting immunization is initiated (FIG. 1B). Inthis respect, the intact Fc portion of the bispecific or trispecificantibodiy is of particular importance mediating the binding to accessorycells such as monocytes/macrophages and dendritic cells and inducingthese cells to become themselves cytotoxic and/or simultaneouslytransfer important co-stimulatory signals to the T cell (FIG. 1B). Inthis manner, it seems to be possible that a T cell reaction may beinduced also against so far unknown tumour-specific peptides.

[0041] Redirection of possibly anergized tumour-specific T cells totumour cells by means of bispecific and/or trispecific antibodies andconcomitant co-stimulation of such T cells by accessory cells bound tothe Fc portion of the bispecific or trispecific antibody might act toreverse the anergy of cytotoxic T cells (CTLs). I.e. using intactheterologous bispecific and/or trispecific antibodies a T cell toleranceexisting in the patient against the tumour may be neutralized and,thereby, a long-lasting anti-tumour immunity may be induced.

[0042] The last aspect is supported by preliminary in vivo data fromexperiments with mice indicating a long-lasting anti-tumour immunityfollowing treatment with a syngeneic tumour and intact bsab. In theseexperiments a total of 14 out of 14 animals which could be successfullytreated with bsab after a first tumour injection survived another tumourinjection 144 days after the first one—without further administration ofbsab (see Example 2).

[0043] Further advantages in the ex vivo immunization by bispecificand/or trispecific antibodies are (i) minimizing possible side effects,(ii) controlled binding of the tumour binding arm to the tumour cellsoutside of the body, and (iii) use of as little bispecific andtrispecific antibodies as possible. Principally, there are two differentways of proceeding which will be detailed in the following. An importantaspect with long-term incubation is that the bispecific or trispecificantibody employed is exhausted and degraded during the incubation periodplanned. In this way, this immunization would avoid the lengthy drugapproval process.

[0044] In the short-term and long-term incubation procedures, the tumourcells are incubated with antibodies over a period of 10 minutes to 5hours, preferably up to 3 hours, further preferred up to 2 hours andstill further preferred 15 minutes to 1 hour. Preferably, the incubationis carried out at a temperature of 4° C. to 25° C., particularlypreferred 4° C. to 10° C. The incubation is preferably performed in asterile environment in bufferd saline having a neutral pH. In the caseof short-term incubation, reinfusion into the patient is performedimmediately afterwards. In the long-term incubation procedure, followingthis preincubation mononucleated peripheral blood cells are added andincubated together with the preincubated tumour cells/antibodies for afurther period of 1 to 14 days, more preferably 3 to 10 days, furtherpreferred 6 to 10 days. Preferably, this incubation is performed at 37°C. under sterile conditions as well as under GMP conditions (GoodManufacturing Production=GMP) in an incubator. As detailed above, inlong-term incubation the blood cells may alternatively be incubatedtogether with tumour cells and antibodies under suitable conditions.

[0045] The incubation conditions described above are only intended to bean example. Depending on the tumour cells and the antibodies used alsoother time periods, temperature conditions etc., and in generaldifferent incubation conditions may be used. By simple experimentation,the skilled artisan will be able to establish such conditions.

[0046] During preincubation the tumour cells are preferably employed inan amount of 10⁷ to 10⁹ cells, further preferred in an amount of about10⁸ cells. The peripheral blood mononucleated cells are added in anamount of about 10⁸ to 10¹⁰ cells. Naturally, the skilled artisan mayselect different incubation conditions which may be determined bylaboratory experimentation (for example changes in cell number andincubation period). The bi-specific and/or tri-specific antibodies usedin the method of the present invention are added in an amount of 2 to100 μg, more preferably 5 to 70 μg, particularly preferred 5 to 50 μg.

[0047] The autologous tumour cells employed are for example irradiatedto prevent further survival of tumour cells. For example, gammaradiation is used e.g. employed in a radiation dose of 50 to 100 Gy. Inanother embodiment of the present invention the autologous tumour cellsare treated by chemical substances, for instance by mitomycin C toprevent their further survival.

[0048] The antibodies used according to the invention are preferablyable to reactivate tumour-specific T cells being in an anergic state.Further, they are able to induce tumour-reactive complement-bindingantibodies and thereby a humoral immune reaction.

[0049] Binding preferably takes place via CD3, CD2, CD4, CD5, CD6, CD8,CD28, and/or CD44 to the T cell. Fc receptor-positive cells at leastbear a Fcγ receptor I, II, or III.

[0050] Antibodies which may be employed according to the invention areable to bind to monocytes, macrophages, dendritic cells, “naturalkiller” cells (NK cells) and/or activated neutrophils being Fcγ receptor1-positive cells.

[0051] The antibodies which may be used according to the invention leadto an induction or increase in the expression of CD40, CD80, CD86,ICAM-1, and/or LFA-3 as co-stimulatory antigens and/or cytokin secretionby the Fc receptor-positive cell. The cytokins preferably are IL-1,IL-2, IL-4, IL-6, IL-8, IL-12, and/or TNF-α.

[0052] Binding to the T cell preferably takes place via the T cellreceptor complex of the T cell.

[0053] The bispecific antibodies which may be used according to theinvention preferably are:

[0054] an anti-CD3 X anti-tumour-associated antigen antibody and/oranti-CD4 X anti-tumour-associated antigen antibody and/or anti-CD5 Xanti-tumour-associated antigen antibody and/or anti-CD6 Xanti-tumour-associated antigen antibody and/or anti-CD8 Xanti-tumour-associated antigen antibody and/or anti-CD2 Xanti-tumour-associated antigen antibody and/or anti-CD28 Xanti-tumour-associated antigen antibody and/or anti-CD44 Xanti-tumour-associated antigen antibody.

[0055] The trispecific antibodies which may be employed according to theinvention preferably are:

[0056] an anti-CD3 X anti-tumour-associated antigen antibody and/oranti-CD4 X anti-tumour-associated antigen antibody and/or anti-CD5 Xanti-tumour-associated antigen antibody and/or anti-CD6 Xanti-tumour-associated antigen antibody and/or anti-CD8 Xanti-tumour-associated antigen antibody and/or anti-CD2 Xanti-tumour-associated antigen antibody and/or anti-CD28 Xanti-tumour-associated antigen antibody and/or anti-CD44 Xanti-tumour-associated antigen antibody.

[0057] The trispecific antibodies useful according to the invention atleast have a T cell binding arm, a tumour cell binding arm and a bindingarm which binds to Fc receptor positive cells. The latter of the bindingarms mentioned may be an anti-Fc receptor binding arm or a mannosereceptor binding arm.

[0058] The bispecific antibody preferably is a heterologous intactrat/mouse bispecific antibody.

[0059] By the bispecific and trispecific antibodies useful according tothe invention T cells are activated and redirected against the tumourcells. Heterologous intact bispecific antibodies which may be preferablyused are selected from one or more of the following isotypecombinations:

[0060] rat-IgG2b/mouse-IgG2a,

[0061] rat-IgG2b/mouse-IgG2b,

[0062] rat-IgG2b/mouse-IgG3;

[0063] rat-IgG2b/human-IgG1,

[0064] rat-IgG2b/human-IgG2,

[0065] rat-IgG2b/human-IgG3[oriental allotype G3m(st)=binding to proteinA],

[0066] rat-IgG2b/human-IgG4;

[0067] rat-IgG2b/rat-IgG2c;

[0068] mouse-IgG2a/human-IgG3[caucasian allotypes G3m(b+g)=no binding toprotein A, in the following indicated as *]

[0069]mouse-IgG2a/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]human-IgG3*-[CH2-CH3]

[0070]mouse-IgG2a/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-humanIgG3*-[CH2-CH3]

[0071]mouse-IgG2a/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0072]mouse-[VH-CH1,VL-CL]-human-IgG1/rat-[VH-CH1,VL-CL]human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0073]mouse-[VH-CH1,VL-CL]-human-IgG4/rat-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4[N-terminalregion of CH2]human-IgG3*[C-terminal region of CH2: >aa position251]-human-IgG3*[CH3]

[0074] rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge-CH2-CH3]

[0075] rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG2-[hinge-CH2-CH3]

[0076] rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG3-[hinge-CH2-CH3,oriental allotype]

[0077] rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG4-[hinge-CH2-CH3]

[0078]human-IgG1/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0079]human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4[N-terminalregion of CH2]-human-IgG3*[C-terminal region of CH2: >aa position251]-human-IgG3*[CH3]

[0080]human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4[N-terminalregion of CH2]-human-IgG3*[C-terminal region of CH2: >aa position251]-human-IgG3*[CH3]human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG2[N-terminalregion of CH2]-human-IgG3*[C-terminal region of CH2: >aa position251]-human-IgG3*[CH3]

[0081]human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG2[N-terminalregion of CH2]-human-IgG3*[C-terminal region of CH2: >aa position251]-human-IgG3*[CH3]

[0082]human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0083]human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0084]human-IgG2/human-[VH-CH1,VL-CL]-human-IgG2-[hinge]-human-IgG3*-[CH2-CH3]

[0085]human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG3*-[CH2-CH3]

[0086]human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4[N-terminalregion of CH2]-human-IgG3*[C-terminal region of CH2: >aa position251]-human-IgG3*[CH3]

[0087]mouse-IgG2b/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0088]mouse-IgG2b/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0089]mouse-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0090]mouse-[VH-CH1,VL-CL]-human-IgG4/rat-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3]

[0091]human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3]

[0092]human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3]

[0093]human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3]

[0094] The antibodies useful according to the invention preferably aremonoclonal, chimeric, recombinant, synthetic, semi-synthetic orchemically modified intact antibodies having for example Fv, Fab, scFvor F(ab)₂ fragments.

[0095] Preferably used are antibodies or derivatives or fragments ofhuman origin or antibodies altered in a way that makes them suitable forapplication to humans (so-called “humanized antibodies”) (see forexample Shalaby et al., J. Exp. Med. 175 (1992), 217; Mocikat et al.,Transplantation 57 (1994), 405).

[0096] The preparation of the various types of antibodies and fragmentsmentioned above is obvious to one skilled in the art. The preparation ofmonoclonal antibodies preferably originating from mammals, e.g. humans,rat, mouse, rabbit or goat, may be performed using conventional methods,as for example described in Köhler and Milstein (Nature 256 (1975),495), in Harlow and Lane (Antibodies, A Laboratory Manual (1988), ColdSpring Harbour) or in Galfré (Meth. Enzymol. 73 (1981), 3).

[0097] Furthermore, it is possible to prepare the antibodies describedby means of recombinant DNA technology according to techniques obviousto the skilled artisan (see Kurucz et al., J. Immunol. 154 (1995), 4576;Hollinger et al., Proc. Natl. Acad. Sc. USA 90 (1993), 6444).

[0098] The antibodies used in the present method can be designed andmanufactured by a person skilled in the art without undue burden. Theenclosed list of references, particularly references (7) to (11)describe methods on how to obtain bispecific and trispecific antibodiesto be used in the present invention.

[0099] Particularly document (9) of Greenwood et al. discloses theexchange of single immunoglobulin domains (for instance CH2) by suitablecloning technique. By using these cloning technique novel antibodycombinations described for instance in claim 9 can be provided. Examplesare:

[0100] human-(VH-CH1, VL-CL)-human IgG4-(hinge)-human IgG4 (N-terminaleregion of CH2)-human IgG3* (C-terminal region of CH2:>aminoacid position251)-human IgG3* (CH3).

[0101] The combination with an antibody: human IgG4 for the preparationof the bispecific antibody: human IgG4/human-(VH-CH1, VL-CL)-humanIgG4-(hinge)-human IgG4 (N-terminal region of CH2)-human IgG3*(C-terminal region of CH2:>aminoacid position 251)-human IgG3* (CH3) isprepared by simple cell fusion as described for instance in document(6).

[0102] On the one hand, the preparation of antibodies having twodifferent specificities, the so-called bispecific antibodies, may beperformed using recombinant DNA technologie, but on the other hand alsoby the so-called hybrid-hydridoma fusion technique (see for exampleMilstein et al., Nature 305 (1983), 537). By this technique, hybridomacell lines producing antibodies each having one of the desiredspecificities are fused, and recombinant cell lines producing antibodieswith both specificities are identified and isolated.

[0103] The problem underlying the invention may be solved both bybispecific and by trispecific antibodies insofar as they show thefeatures and activities characterized in claim 1. In the following, thepreparation of antibodies having two and three specificities isdescribed in more detail. To provide such bispecific and trispecificantibodies belongs to the state of the art, and the literaturedescribing such methods of preparation is hereby incorporated byreference in its entirety.

[0104] The preparation of antibodies having three specificities,so-called trispecific antibodies, which are also suitable to solve thefundamental problem of the invention may be for example carried out bycoupling to one of the heavy IgG chains of a bispecific antibody a thirdantigen-binding site having another specificity, e.g. in the form of“single chain variable fragments” (scFv). The scFv may be for examplebound to one of the heavy chains via a

-S-S(G₄S)_(n)D-I linker

[0105] (S=serine, G=glycine, D=aspartate, I=isoleucine).

[0106] Analogously, trispecific F(ab)₂ constructs may be preparedsubstituting the CH2-CH3 regions of the heavy chain of one specificityof a bispecific antibody by a scFv of a third specificity while theCH2-CH3 regions of the heavy chain of the other specificity are removed,e.g. by introduction of a stop codon (at the end of the “hinge” region)into the coding gene for example by homologous recombination (see FIG.5).

[0107] It is also possible to prepare trispecific scFv constructs. Inthis case three VH-VL regions representing three different specificitiesare arranged in series (FIG. 6).

[0108] According to the invention, there are for example used intactbispecific antibodies. Intact bispecific antibodies are a combination oftwo antibody semi-molecules (each of one H and L immunoglobulin chain)each representing one specificity and, like normal antibodies, having inaddition a Fc portion which performs the well known effector functions.Preferably, they are prepared by quadroma technology. This method ofpreparation is described representatively in DE-A-44 19 399. Thisdocument is incorporated by reference in its entirety for the purpose ofcomplete diclosure also with respect to a definition of bispecificantibodies. Naturally, also other methods of preparation may be employedas long as they result in the intact bispecific antibodies defined aboverequired according to the invention.

[0109] For example, by a newly developed method of production (6) intactbispecific antibodies may be prepared in a sufficient amount. Thecombination of 2 bispecific antibodies against 2 differenttumour-associated antigens (e.g. c-erb-B2, ep-cam, such asGA-733-2=C215) on the mamma carcinoma cells minimizes the risk thattumour cells expressing only one antigen are not recognized.

[0110] There have also been attempts to achieve an anti-tumour immunityby treatment with bispecific F(ab′)2 fragments having the specificitiesof anti-c-erb-B2 x anti CD64. The main disadvantage of bsF(ab′)2fragments is that due to the specificities used only FcγRI+ cells areredirected to the tumour. T cells are not redirected to the tumour bythis bispecific antibody. While bsF(ab′)2 fragments have the potenntialto directly destroy the tumour, they are unable to establish ananti-tumour immunity themselves. Only the T cell with its specific Tcell receptor has this capability. While the FcγRI+ cells are able toindirectly activate tumour-specific T cells by presentingtumour-specific peptides (via MHCI or MHCII, respectively), for examplefollowing phagocytosis of tumour components, the efficiency of inductionof an anti-tumour immunity is this case is not as high (only in 30% ofthe patients).

[0111] Further advantages of intact bsabs capable of redirecting T cellsas compared to the above-mentioned bsF(ab′)2 fragments are detailed inthe following:

[0112] 1. To intact bsabs there may bind Fc receptor-positive cells andmay on the one hand by ADCC (antibody-dependent cell-mediatedcytotoxicity) contribute directly to the destruction of the tumour andon the other hand to T cell activation, as detailed above.

[0113] 2. By intact T cell-redirecting bsabs also energizedtumour-specific T cells are directed to the tumour cell which accordingto the invention may be directly reactivated at the tumour. This may notbe achieved using a bsF(ab′)2 fragment having the specificities of antiCD64 x anti tumour-associated antigen.

[0114] 3. A bsF(ab′)2 fragment having the specificities of anti CD64 xanti tumour-associated antigen is merely able of achieving ananti-tumour immunity in 30% of the patients while according to theinvention in experiments with mice using T cell-redirecting intact bsabsa protection in 100% of the animals could be achieved.

[0115] Binding of the bsabs to Fcγ-RI has two significant advantageswith respect to optimum anti-tumour effectivity:

[0116] (1) Fcγ-RI-positive cells are capable of eliminating tumour cellsby means of ADCC (11) and in this respect may contribute synergisticallyto the anti-tumour-effect of the cytotoxic T cells which have beendirected to the tumour cell by the bsab (13).

[0117] (2) Fcγ-RI-positive cells (such asmonocytes/macrophages/dendrites) are capable of providing importantco-stimulatory signals similar to antigen presentation to the T cell andthereby to prevent energizing of the T cell. Furthermore, as shown inFIG. 1, as a desired side product due to the intact bsab-mediatedinteraction of the T cell with accessory cell and tumour cell there maybe stimulated T cells having a T cell receptor which recognizestumour-specific peptides (presented on the tumour cell via MHCantigens). The co-stimuli necessary for a correct activation of the Tcell in this situation would be provided by the accessory cell (e.g. themonocyte). In this respect, the antibody of the invention besides thedirect T cell receptor-independent bsab-mediated tumour destruction(FIG. 1A) should also activate and generate tumour-specific T cells(FIG. 1B) which after degradation of the bsabs continue to patrol in thepatient. I.e. by means of intact bsabs similar to genetherapeuticalapproaches (e.g. by incorporation of co-stimulatory antigens such as B-7into the tumour cell) the tumour tolerance in the patient may beovercome.

[0118] In this respect, it is further beneficial that the expression ofFcγ-RI is up-regulated on the respective cells following G-CSFtreatment.

[0119] The invention has been described in the above and will bedescribed in the following in particular with respect to bispecificantibodies. Instead of bispecific antibody, of course also trispecificantibodies may be used as long as they comply with the provisions made.

[0120] The invention has been and will be described with respect to theaccompanying Figures. The Figures show:

[0121]FIG. 1: the role of accessory cells in tumour immunotherapy bymeans of bispecific antibodies;

[0122]FIG. 2: the destruction of tumour cells following administrationof bispecific antibodies as evidenced by flow-cytometry;

[0123]FIG. 3: induction of cytokins by intact bispecific antibodies onlybut not by parental antibodies;

[0124]FIG. 4: efficiency of the method according to the invention invivo;

[0125]FIG. 5: trispecific F(ab)₂ antibodies;

[0126]FIG. 6: trispecific scFv antibody.

IMMUNIZATION PROTOCOLS

[0127] Ex vivo Immunization (Short-term Incubation)

[0128] 1. Preparation of a single cell suspension (10⁷-10⁹ cells) fromautologous tumour material (or allogenic tumour cells of the same tumourtype) with subsequent γ irradiation (50-100 Gy).

[0129] 2. Addition of bsabs (5-50 μg) and incubation for 45 minutes at4° C. Afterwards washing away of unbound antibodies.

[0130] 3. Reinfusion of the cell mixture (i.v.).

[0131] Ex vivo Immunization (Long-term Incubation)

[0132] 1. Preparation of a single cell suspension (10⁷-10⁹ cells) fromautologous tumour material (or allogenic tumour cells of the same tumourtype) with subsequent γ irradiation (50-100 Gy).

[0133] 2. Addition of bsabs (5-50 μg), 45 minutes incubation.

[0134] 3. Addition of PBMCs (10⁸-10¹⁰), [alternatively: 1×10⁹ cellsobtained from T cell aphaeresis].

[0135] 4. After 5 to 7 days monitoring of T cell reactivity by transferof aliquots e.g. to allogenic breast cancer cell lines (MCF-7, MX-1).

[0136] 5. Reinfusion (i.v.) of the cultured PBMCs on days 4 to 14 intothe patient (in the case of T cell aphaeresis: cryo conservation).

[0137] Abbreviations: PBMCs, peripheral blood mononucleated cells; i.v.,intravenously.

[0138] A similar assay but instead depending on the addition of cytokinsand carried out using conventional bsabs (no activation of accessorycells by bsabs of the subclass combination rat IgG2B x rat IgG1)demonstrates the principal effectivity of such an ex vivo immunizationin the animal model (5).

[0139] In contrast to this, the advantage of the method disclosed hereinresides in the “self-sufficiency” with respect to cytokins (such asINF-α or TNF-α) required for an up-regulation of for example MHC 1 onthe tumour cell by simultaneous activation of T cells and accessorycells (monocytes/macrophages, Fig.) on the tumour cell. This is achievedby the particular subclass combination mentioned at the beginning of theintact bsab used herein. In the case of short-term incubation theseprocesses take place in the patient. Further advantages in short-termincubation are (i) avoiding the cultivation of the cell suspension withserum-containing medium otherwise necessary. (ii) Due to this, also thecost-intensive cultivation according to GMP regulations may be ommitted.(iii) A further important aspect is avoidance or reduction,respectively, of possible side effects by the bsab because of thesignificantly lower amount of antibodies applied.

[0140] An advantage in long-term incubation is that the bsab in vitroafter some time exhausts itself (and, thus, this method may beestablished not as a medicament but as a “medical device”).

EXAMPLE 1

[0141] Bispecific Antibody-mediated Lysis of Tumour Cells by Allogenic TCells

[0142] H-Lac78 is a cell line which has been established from ahypopharynx carcinoma and which expresses epcam to a high extent (ownFACS data). Using H-Lac78 and peripheral mononucleated cells (PBMC) fromvolunteers it was possible to detect the generation of allogeniccytotoxic T lymphocytes. For this purpose, constant amounts of H-Lac78(2×10⁴) were incubated with varying amounts of PBMCs in the presence (10ng) or absence of a bsab (anti epcam x anti CD3). After a period ofseven days the PBMCs were removed and analysed in a flow-cytometer. Atthe same time, the number of H-Lac78 tumour cells was determined. Theactivation of T cells may be observed microscopically by means ofcluster formation; proliferation may be evidenced by the incorporationof radiolabeled thymidine. The-detection of remaining tumour cells isperformed microscopically as well as by the epithelial marker epcamwhich is not expressed on peripheral blood cells. As shown in FIG. 2,the H-Lac78 cells were completely lysed in the presence of bsab, i.e. noepcam-positive cells were detectable in the flow-cytometer after sevendays. These data were confirmed by microscopic oberservations. Incontrast, without bsab a confluent layer of H-Lac78 cells was observedin the wells and epcam-positive cells were detectable by FACS.

[0143] Detection of Activated Allospecific CTLs by Transfer Experiment

[0144] In a subsequent transfer experiment the PBMCs incubated with orwithout bsab, respectively, were transferred onto new H-Lac78 cellswithout readdition of bsab. Also in this case, the tumour cells werelysed but exclusively by PBMCs which had been activated by bsabpreviously. H-Lac78 lysis was complete within 24 hours up to a ratio of2 PBMCs to 1 H-Lac78 cell. This result indicates the generation ofallospecific CTLs without external addition of interleukin-2 (IL-2).Since IL-2 is essential for the activation of T lymphocytes, the dataobtained herein suggest that by bsab-mediated activation IL-2 isproduced by the T cells themselves. Induction of IL-2 mRNA by additionof bsab could be confirmed afterwards by RT-PCR where the bsab wasclearly superior to the parental starting antibodies (FIG. 3). Thisobservation is important insofar as IL-2 has been described as ananti-tumour effective cytokin; but the systemic administration of whichin an appropriate concentration is limited because of its toxicity. Incontrast, the risk of toxicity does not appear in the local productionof IL-2 as it is for example induced by intact bsab. Also, since aneffective induction of IL-2 (and IL-12) requires stimulation of T cellsvia the T cell receptor and CD28, this indicates the importance of Fcreceptor-positive cells (providing the ligands for CD28, CD80, and CD86)in T cell activation by intact bsab.

[0145] BEISPIEL 2

[0146] To address the question whether bispecific antibodies are able toinduce a long-lasting anti-tumour immunity C57BL/6 mice were firstinjected with 5×10³ syngeneic B16 tumour cells. Two days later, a groupof mice (number of 18) were treated with intact bsab prepared byquadroma technology (6) and recognizing a target structure(ep-cam/C215=tumour-associated antigen) on the tumour cell as well asCD3 on the T cells. A second group (number of 6) received an equimolaramount of Fab fragments of both of the specificities contained in thebsab only. While all of the animals of the Fab control group died or hadto be sacrificed within 56 days, 14 of the 18 animals treated with bsabsurvived. 144 days after the first injection of tumour cells the 14surviving animals were injected with another dose of 750 B16 tumourcells but this time without administration of bsabs. As a control, thesame number of tumour cells was administered to 5 untreated animals.While the last animal of the untreated control group had to besacrificed 66 days after tumour injection, all of the animals treatedwith bsab survived (monitoring period: 120 days following second tumourcell injection). See also FIGS. 4A and B: Survival graphs of the twosubsequent experiments described above.

1. Method for ex vivo immunization of humans and animals comprising thefollowing steps of: a) isolating autologous tumour cells; b) treatingthe tumour cells to prevent the survival thereof following reinfusion;c) incubating the thus treated tumour cells with intact heterologousbispecific and/or trispecific antibodies showing the followingproperties: α—binding to a T cell; β—binding to at least one antigen ona tumour cell; γ—binding, by their Fc portion (in the case of bispecificantibodies), or by a third specificity (in the case of trispecificantibodies) to Fc receptor-positive cells.
 2. Method according to claim1, characterized in that said antibodies are selected so that they arecapable of binding Fc receptor-positive cells having a Fcγ receptor I,II, or III.
 3. Method according to claim 2, characterized in that saidantibodies are capable of binding to monocytes, macrophages, dendriticcells, “natural killer” cells (NK cells) and/or activated neutrophilsbeing Fcγ receptor I-positive cells.
 4. Method according to claim 1,characterized in that said antibodies are capable of inducingtumour-reactive complement-binding antibodies and thus inducing ahumoral immune response.
 5. Method according to claim 1, characterizedin that said antibodies are selected to bind to the T cells via CD2,CD3, CD4, CD5, CD6, CD8, CD28 and/or CD44.
 6. Method according to claim1, characterized in that said antibodies are selected so that followingtheir binding to the Fc receptor-positive cells the expression of CD40,CD80, CD86, ICAM-1 and/or LFA-3 as co-stimulatory antigens, and/orsecretion of cytokins by the Fc receptor-positive cell is initiated orincreased.
 7. Method according to claim 6, characterized in that saidantibodies are selected so that the secretion of IL-1, IL-2, IL-4, IL-6,IL-8, IL-12 being cytokins and/or of TNF-α is increased.
 8. Methodaccording to claim 1, characterized in that said bispecific antibody isselected to be an anti-CD3 X anti-tumour-associated antigen antibodyand/or anti-CD4 X anti-tumour-associated antigen antibody and/oranti-CD5 X anti-tumour-associated antigen antibody and/or anti-CD6 Xanti-tumour-associated antigen antibody and/or anti-CD8 Xanti-tumour-associated antigen antibody and/or anti-CD2 Xanti-tumour-associated antigen antibody and/or anti-CD28 Xanti-tumour-associated antigen antibody and/or anti-CD44 Xanti-tumour-associated antigen antibody.
 9. Method according to one ormore of the preceding claims, characterized in that said bispecificantibody is selected from one or more of the following isotypecombinations: rat-IgG2b/mouse-IgG2a, rat-IgG2b/mouse-IgG2b,rat-IgG2b/mouse-IgG3; rat-IgG2b/human-IgG1, rat-IgG2b/human-IgG2,rat-IgG2b/human-IgG3[oriental allotype G3m(st)=binding to protein A],rat-IgG2b/human-IgG4; rat-IgG2b/rat-IgG2c;mouse-IgG2a/human-IgG3[caucasian allotypes G3m(b+g)=no binding toprotein A, in the following indicated as *]mouse-IgG2a/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]mouse-IgG2a/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]mouse-IgG2a/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]mouse-[VH-CH1,VL-CL]-human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]mouse-[VH-CH1,VL-CL]-human-IgG4/rat-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4[N-terminalregion of CH2]-human-IgG3*[C-terminal region of CH2: >aa position251]-human-IgG3*[CH3]rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge-CH2-CH3]rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG2-[hinge-CH2-CH3]rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG3-[hinge-CH2-CH3, orientalallotype] rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG4-[hinge-CH2-CH3]human-IgG1/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]human-IgG3*-[CH2-CH3]human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4[N-terminalregion of CH2]-human-IgG3*[C-terminal region of CH2: >aa position251]-human-IgG3*[CH3]human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4[N-terminalregion of CH2]-human-IgG3*[C-terminal region of CH2: >aa position251]-human-IgG3*[CH3]human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG2[N-terminalregion of CH2]-human-IgG3*[C-terminal region of CH2: >aa position251]-human-IgG3*[CH3]human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG2[N-terminalregion of CH2]-human-IgG3*[C-terminal region of CH2: >aa position251]-human-IgG3*[CH3]human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]human-IgG2/human-[VH-CH1,VL-CL]-human-IgG2-[hinge]-human-IgG3*-[CH2-CH3]human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG3*-[CH2-CH3]human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4[N-terminalregion of CH2]-human-IgG3*[C-terminal region of CH2: >aa position251]-human-IgG3*[CH3]mouse-IgG2b/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]mouse-IgG2b/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]mouse-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]mouse-[VH-CH1,VL-CL]-human-IgG4/rat-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3]human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3]human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3]human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3]10. Method according to claim 1, characterized in that said bispecificantibody is selected from a heterologous rat/mouse bispecific antibody.11. Method according to claim 1, characterized in that said trispecificantibody has a T cell binding arm, a tumour cell binding arm and a thirdspecificity for binding to Fc receptor-positive cells.
 12. Methodaccording to claim 11, characterized in that said trispecific antibodyis selected to be an anti-CD3 X anti-tumour-associated antigen antibodyand/or anti-CD4 X anti-tumour-associated antigen antibody and/oranti-CD5 X anti-tumour-associated antigen antibody and/or anti-CD6 Xanti-tumour-associated antigen antibody and/or anti-CD8 Xanti-tumour-associated antigen antibody and/or anti-CD2 Xanti-tumour-associated antigen antibody and/or anti-CD28 Xanti-tumour-associated antigen antibody and/or anti-CD44 Xanti-tumour-associated antigen antibody.
 13. Method according to claim1, characterized in that in said step c) after incubating the tumourcells with intact heterologous bispecific and/or trispecific antibodiesthe tumour cells charged with antibodies are prepared for reinfusion(short-term incubation).
 14. Method according to claim 1, characterizedin that in said step c) the incubation of the tumour cells withantibodies is performed together with mononucleated cells of theperipheral blood (PBMC=peripheral blood mononucleated cells), ormononucleated cells are added after incubation of the tumour cells withthe antibodies and the incubation is continued (long-term incubation).15. Method according to claim 13 or 14, characterized in that saidtumour cells are incubated with the antibodies for a period of 10minutes to 5 hours.
 16. Method according to claim 13 or 14,characterized in that said tumour cells are incubated with theantibodies for a period of 15 minutes to 120 minutes.
 17. Methodaccording to claim 14, characterized in that said mononucleatedperipheral blood cells are incubated with the tumour cells and theantibodies for a period of 1 to 14 days.
 18. Method according to claim14, characterized in that said mononucleated peripheral blood cells areadded in an amount of about 10⁸ to 10¹⁰ cells.
 19. Method according toclaim 1, characterized in that said tumour cells are added in an amountof 10⁷ to 10⁹ cells.
 20. Method according to claim 1, characterized inthat said bispecific and/or trispecific antibodies are added in anamount of 2 to 100 μg.
 21. Method according to claim 1, characterized inthat said treating of the tumour cells in step b is performed byirradiation.
 22. Method according to claim 1, characterized in that,said bispecific and/or trispecific antibodies are capable of activatingthe Fc receptor-positive cell whereby the expression of cytokins and/orco-stimulatory antigens is induced or increased.
 23. Use of the tumourcell containing preparation according to claim 1 or 14 in the preventionand treatment of tumourous diseases.
 24. Use according to claim 23 forinducing an anti-tumour immunity.
 25. Method according to claim 1 forthe preparation of autologous tumour cells treated with heterologousbispecfic and/or trispecific antibodies for reinfusion into the patientor the animals from whom the autologous tumour cells have been obtained.26. A pharmaceutical composition containing a tumour cell preparationobtained by the method of claim 1 or 14.